We began our work to discover genes required for expression or function of fusion oncogenes by using the results of a genome-wide RNAi screen designed to identify genes needed for the activity of the fusion oncoprotein EWS-FLI1. The development of this RNAi screen was a collaborative effort involving my laboratory and researchers from CCR's Pediatric Oncology Branch and the trans-NIH RNAi screening facility at the National Center for Advancing Translational Sciences (NCATS). EWS-FLI1 is an oncogenic transcription factor that results from a chromosomal rearrangement involving chromosomes 11 and 22. The chromosome 11:22 translocation is the most frequent primary genetic event observed in Ewing sarcoma (EWS), a cancer of the bone and soft tissues. The genome-wide RNAi screen revealed the expression of the EWS-FLI1 fusion transcript is sensitive to the inhibition of the activity of specific splicing factors. We identified that expression of the EWS-FLI1 transcripts expressed in ES cells harboring translocations where the breakpoint in chromosome 22 occurs within a region of DNA (intron 8 of the EWSR1 gene) requires the splicing factor HNRNPH1 to generate an in-frame mRNA. The depletion of HNRNPH1 in EWS cells that harbor a chromosome 22 breakpoint in EWSR1 intron 8, disrupts the expression and the activity of the EWS-FLI1 protein and reduces cell survival. Ongoing studies are investigating how HNRNPH1 facilitates the splicing of EWS-FLI1 in those EWS cells that depend upon its function, with the long-term aim of identifying means of inhibiting this event. Follow up studies of the genome-wide RNAi screen of EWS-FLI1 activity also identified SF3B1, the catalytic component of the spliceosome, a protein complex required for splicing, as a protein that EWS cells are particularly dependent upon for cell survival. We discovered that the biogenesis of the EWS-FLI1 mRNA is sensitive to disruption of spliceosome function and that canonical splicing is a potential vulnerability in EWS. We are currently extending our analysis to other components of the spliceosome, and we are assessing new methods for investigating the splicing of fusion oncogenes. Transcription factors have long been considered viable therapeutic targets for cancer. However, inhibiting transcription factor function using small molecules has proven challenging. A potential alternative strategy is the targeting of post-translational modifications of proteins. Previous studies have reported post-translational modifications of the EWS-FLI1 protein, but we know little about the proteins that catalyze these changes. We are thus using functional genetic approaches to identify candidate proteins required for the post-translational modification of the EWS-FLI1 protein, particularly proteins that may regulate protein stability. We are also investigating the reprogramming of the transcriptome and signaling networks by the EWS-FLI1 fusion oncoprotein that suppresses the differentiation of the progenitor cells in which EWS arises and promotes tumor growth. For example, we demonstrated recently that EWS-FLI1 positively regulates the expression of proteins required for serine-glycine biosynthesis and uptake of the alternative nutrient source glutamine. Specifically, we show that EWS-FLI1 can alter the expression of PHGDH, PSAT1, PSPH, and SHMT2, genes that encoding enzymes required for serine-glycine biosynthesis. Using cell-based studies, we also established that EWS cells are dependent on glutamine for cell survival and that EWS-FLI1 positively regulates expression of the glutamine transporter, SLC1A5 and two enzymes involved in the one-carbon cycle, MTHFD2 and MTHFD1L. Inhibition of serine-glycine biosynthesis in EWS cells impacts their redox state leading to an accumulation of reactive oxygen species, DNA damage, and apoptosis. Importantly, analysis of EWS primary tumor transcriptome data confirmed that most of the genes identified by our cell-based studies, also exhibit increased expression compared with normal tissues, including PHGDH and SHMT2. Current studies are examining other cellular processes EWS-FLI1 regulates to favor EWS tumorigenesis, including those that support cell proliferation or metastasis.